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PubMed2000

In vitro engineering of human stratified urothelium: analysis of its morphology and function.

PURPOSE: Gastric or intestinal patches, commonly used for reconstructive cystoplasty, may induce severe metabolic complications. The use of bladder tissues reconstructed in vitro could avoid these complications. We compared cellular differentiation and permeability characteristics of human native with in vitro cultured stratified urothelium. MATERIALS AND METHODS: Human stratified urothelium was induced in vitro. Morphology was studied with light and electron microscopy and expression of key cellular proteins was assessed using immunohistochemistry. Permeability coefficients were determined by measuring water, urea, ammonia and proton fluxes across the urothelium. RESULTS: As in native urothelium the stratified urothelial construct consisted of basal membrane and basal, intermediate and superficial cell layers. The apical membrane of superficial cells formed villi and glycocalices, and tight junctions and desmosomes were developed. Immunohistochemistry showed similarities and differences in the expression of cytokeratins, integrin and cellular adhesion proteins. In the cultured urothelium cytokeratin 20 and integrin subunits alpha6 and beta4 were absent, and symplekin was expressed diffusely in all layers. Uroplakins were clearly expressed in the superficial umbrella cells of the urothelial constructs, however, they were also present in intermediate and basal cells. Symplekin and uroplakins were expressed only in the superficial cells of native bladder tissue. The urothelial constructs showed excellent viability, and functionally their permeabilities for water, urea and ammonia were no different from those measured in native human urothelium. Proton permeability was even lower in the constructs compared to that of native urothelium. CONCLUSIONS: Although the in vitro cultured human stratified urothelium did not show complete terminal differentiation of its superficial cells, it retained the same barrier characteristics against the principal urine components. These results indicate that such in vitro cultured urothelium, after being grown on a compliant degradable support or in coculture with smooth muscle cells, is suitable for reconstructive cystoplasty.

Cell Differentiation↗
PubMed1999

Localization and expression of integrin subunits in the embryoid bodies of F9 teratocarcinoma cells.

F9 embryonal carcinoma cells can differentiate in vitro into either parietal (PE) or visceral (VE) endoderm, depending upon specific retinoic acid (RA) treatment and growth conditions. In differentiated aggregates of F9 cells (EB), the VE is a polarized monolayer surrounding a core of undifferentiated cells. Within 7 days of treatment the cells organize their cytoskeleton and synthesize large amounts of extracellular matrix proteins to form a basal lamina under the newly formed epithelium. All these changes are likely to involve integrin expression and organization. In this study we have analyzed the spatio-temporal changes in the pattern and level of expression of beta1, beta4, alpha5, alpha6A, and alpha6B integrin subunits. We found that the organization of the VE monolayer in F9 aggregates involves both qualitative and quantitative changes in integrin expression. beta1 is downregulated and accumulates in the forming epithelium. The same occurs for alpha5, although its location on the surface of the aggregate appears to be transient as in fully differentiated EB its distribution is uniform. beta4 and alpha6A are also mainly localized in the VE but they are undetectable in undifferentiated aggregates and their expression is induced by RA treatment. An important exception is represented by alpha6B whose distribution and expression remain almost unchanged throughout treatment.

Animals↗
PubMed1999

Acquired skin disease of hemidesmosomes.

The hemidesmosome is a membrane-associated supramolecular dermal epidermal complex linking the cytoskeleton of the basal keratinocyte to structures within the papillary dermis. Different components of this complex have been identified as autoantigens in autoimmune bullous skin diseases. Some of the autoantigens have been characterized at the molecular level. Little is known, however, about the factors that initiate the production of autoantibodies. By histopathology, acquired skin diseases of hemidesmosomes show subepidermal blisters and by direct immunofluorescence, linear deposits of IgG, C3 or IgA at the dermal epidermal junction. Bullous pemphigoid (BP) is the most common acquired disease of hemidesmosomes. Two proteins, BP180 and BP230, have been identified as primary targets of autoantibodies in BP. In addition, pemphigoid/herpes gestationis, lichen planus pemphigoides, cicatricial pemphigoid and linear IgA disease are characterized by an immune response to BP180. Laminin 5 is another well-characterized anchoring filament-lamina densa component of hemidesmosomes. Patients with autoantibodies to laminin 5 show the clinical phenotype of cicatricial pemphigoid. Other acquired skin diseases of the hemidesmosomes reveal autoantibodies to a plectin-like protein, the beta4 subunit of alpha6beta4 integrin, uncein and a not yet characterized 168 kDa protein. Recently, diseases with autoantibodies to 105 and 200 kDa proteins of the lower lamina lucida have been reported. The association of these autoantigens with hemidesmosomes still needs to be demonstrated. Finally, anchoring fibrils associate with the dermal epidermal anchoring complex. The major structural component of anchoring fibrils is type VII collagen, the autoantigen of epidermolysis bullosa acquisita.

Animals↗
PubMed1999

Pretreatment of human keratinocyte sheets with laminin 5 improves their grafting efficiency.

Laminin 5 is essential in epithelial attachment to stromal tissues, suggesting that it might improve keratinocyte attachment in a variety of clinical situations. In this study, we examined the effect of exogenous laminin 5 upon the efficiency of transplantation of keratinocyte sheets in animal models. Keratinocyte sheets were prepared according to the method of Rheinwald & Green (1975). Purified laminin 5 was added to the sheets of group 1 (1.0 microg per cm2), Dulbecco's modified Eagle's medium alone was added to group 2. The sheets were grafted to the panniculus carnosus of nude mice (BALB/C nu/nu) (n = 12) and nude rats (Fisher 344) (n = 15). The take rate was assessed by measurement of the area of surviving epithelium at 7 d postgrafting. Laminin 5 bound the keratinocyte sheets of group 1. At 7 d postgrafting, the area of epithelialization of group 1 was significantly larger than that of group 2. Immunohistochemistry staining showed that collagen IV, laminin 5, and collagen VII stained more strongly at the dermal-epidermal junction in group 1 than in group 2. Integrin chains alpha6 and beta4 were similar in both groups. Electron microscopy at day 3 after grafting, showed the lamina densa of group 1 to be more continuous than in group 2. Pretreatment of cultured human keratinocyte sheets with laminin 5 improved the extent of epithelial coverage and increased the rate of neobasement membrane formation. The results suggest that laminin 5 promotes epithelial attachment by increasing the rate of basement membrane assembly.

Animals↗
PubMed1999

PETA-3/CD151, a member of the transmembrane 4 superfamily, is localised to the plasma membrane and endocytic system of endothelial cells, associates with multiple integrins and modulates cell function.

The Transmembrane 4 Superfamily member, PETA-3/CD151, is ubiquitously expressed by endothelial cells in vivo. In cultured human umbilical vein endothelial cells PETA-3 is present on the plasma membrane and predominantly localises to regions of cell-cell contact. Additionally, this protein is abundant within an intracellular compartment which accounts for up to 66% of the total PETA-3 expressed. Intracellular PETA-3 showed colocalisation with transferrin receptor and CD63 suggesting an endosomal/lysosomal localisation which was supported by immuno-electronmicroscopy studies. Co-immunoprecipitation experiments investigating possible interactions of PETA-3 with other molecules demonstrated associations with several integrin chains including beta1, beta3, beta4, (alpha)2, (alpha)3, (alpha)5, (alpha)6 and provide the first report of Transmembrane 4 Superfamily association with the (alpha)6beta4 integrin. Using 2-colour confocal microscopy, we demonstrated similar localisation of PETA-3 and integrin chains within cytoplasmic vesicles and endothelial cell junctions. In order to assess the functional implications of PETA-3/integrin associations, the effect of anti-PETA-3 antibodies on endothelial function was examined. Anti-PETA-3 mAb inhibited endothelial cell migration and modulated in vitro angiogenesis, but had no detectable effect on neutrophil transendothelial migration. The broad range of integrin associations and the presence of PETA-3 with integrins both on the plasma membrane and within intracellular vesicles, suggests a primary role for PETA-3 in regulating integrin trafficking and/or function.

Antigens, CD↗
PubMed2006

Regulation of human neural precursor cells by laminin and integrins.

Deciphering the factors that regulate human neural stem cells will greatly aid in their use as models of development and as therapeutic agents. The extracellular matrix (ECM) is a component of stem cell niches in vivo and regulates multiple functions in diverse cell types, yet little is known about its effects on human neural stem/precursor cells (NSPCs). We therefore plated human NSPCs on four different substrates (poly-L-ornithine, fibronectin, laminin, and matrigel) and compared their responses with those of mouse NSPCs. Compared with the other substrates, laminin matrices enhanced NSPC migration, expansion, differentiation into neurons and astrocytes, and elongation of neurites from NSPC-derived neurons. Laminin had a similar spectrum of effects on both human and mouse cells, highlighting the evolutionary conservation of NSPC regulation by this component of the ECM. Flow cytometry revealed that human NSPCs express on their cell surfaces the laminin-binding integrins alpha3, alpha6, alpha7, beta1, and beta4, and function-blocking antibodies to the alpha6 subunit confirmed a role for integrins in laminin-dependent migration of human NSPCs. These results define laminin and its integrin receptors as key regulators of human NSPCs.

Animals↗
PubMed2006

Slug regulates integrin expression and cell proliferation in human epidermal keratinocytes.

The human epidermis is a self-renewing epithelial tissue composed of several layers of keratinocytes. Within the epidermis there exists a complex array of cell adhesion structures, and many of the cellular events within the epidermis (differentiation, proliferation, and migration) require that these adhesion structures be remodeled. The link between cell adhesion, proliferation, and differentiation within the epidermis is well established, and in particular, there is strong evidence to link the process of terminal differentiation to integrin adhesion molecule expression and function. In this paper, we have analyzed the role of a transcriptional repressor called Slug in the regulation of adhesion molecule expression and function in epidermal keratinocytes. We report that activation of Slug, which is expressed predominantly in the basal layer of the epidermis, results in down-regulation of a number of cell adhesion molecules, including E-cadherin, and several integrins, including alpha3, beta1, and beta4. We demonstrate that Slug binds to the alpha3 promoter and that repression of alpha3 transcription by Slug is dependent on an E-box sequence within the promoter. This reduction in integrin expression is reflected in decreased cell adhesion to fibronectin and laminin-5. Despite the reduction in integrin expression and function, we do not observe any increase in differentiation. We do, however, find that activation of Slug results in a significant reduction in keratinocyte proliferation.

Base Sequence↗
PubMed2006

Thymosin beta4 triggers an epithelial-mesenchymal transition in colorectal carcinoma by upregulating integrin-linked kinase.

The epithelial-mesenchymal transition (EMT) is crucial for the invasion and metastasis of many epithelial tumors including colorectal carcinoma (CRC). In the present study, a scattering and fibroblastic morphology with reduced intercellular contacts was found in the SW480 colon cancer cells overexpressing the gene encoding thymosin beta4 (Tbeta4), which was accompanied by a loss of E-cadherin as well as a cytosolic accumulation of beta-catenin, two most prominent markers of EMT. Whereas E-cadherin downregulation was likely to be accounted by a ZEB1-mediated transcriptional repression, the accumulation of beta-catenin was a result of glycogen synthase kinase-3beta inactivation mediated by integrin-linked kinase (ILK) and/or its downstream effector, Akt. Intriguingly, ILK upregulation in Tbeta4-overexpressing SW480 cells seemed to be attributed mainly to a stabilization of this kinase by complexing with particularly interesting new Cys-His protein (PINCH) more efficiently. In the meantime, a strong correlation between the expression levels of Tbeta4, ILK and E-cadherin in CRC patients was also revealed by immunohistochemical analysis. Taken together, these data suggest a novel role of Tbeta4 in promoting CRC progression by inducing an EMT in tumor cells via upregulating ILK and consequentially its signal transduction.

Adaptor Proteins, Signal Transducing↗
PubMed2003

Rapid decay of alpha6 integrin caused by a mis-sense mutation in the propeller domain results in severe junctional epidermolysis bullosa with pyloric atresia.

Genetic mutations in alpha6beta4 integrin cause junctional epidermolysis bullosa with pyloric atresia, a genodermatosis characterized by blistering of the skin and pyloric occlusion. The lethal form of junctional epidermolysis bullosa with pyloric atresia has been mainly associated with the presence of premature termination codons in the mRNA encoding either the alpha6 or beta4 subunit causing rapid decay of the mutated transcript and absence of alpha6beta4 integrin. In this study, we disclose the genetic background of lethal junctional epidermolysis bullosa with pyloric atresia in a patient presenting absent expression of alpha6 integrin despite normal steady-state level of the alpha6beta4 mRNA. Screening for mutation in the alpha6 gene detected a homozygous base pair substitution (286 C-to-T), which results in the substitution of a serine with a leucine residue (S47L). The amino acid substitution S47L localizes in the first beta-strand of the seven-bladed beta-propeller structure of the extracellular head of alpha6 integrin, and triggers a rapid proteolysis of the aberrant polypeptides involving the lysosomal degradation pathway. This study provides new insight into the pathogenic effect of a mis-sense mutation affecting a functional domain of a protein, and identifies a critical peptide sequence of the beta-propeller domain conserved among the alpha integrin cell receptors.

Amino Acid Sequence↗
PubMed2003

Disruption of the integrity of human peritoneal mesothelium by interleukin-1beta and tumor necrosis factor-alpha.

Inflammatory and neoplastic disease processes of the abdominal cavity are frequently associated with disruption of the integrity of the peritoneal mesothelium. In the present study, we analyzed the effects of the pro-inflammatory cytokines interleukin-1beta (IL-1beta) and tumor necrosis factor-alpha (TNF-alpha) on the morphology and expression of adhesion molecules of human peritoneal mesothelial cells (HPMC). Treatment of HPMC with IL-1beta and TNF-alpha resulted in a time- and dose-dependent alteration of the normal cobblestone morphology of the mesothelium with loss of polarization, cellular retraction and exposure of the submesothelial matrix. The effect was already observable after 6 h of treatment and was most pronounced at a dose of 10 ng/ml of IL-1beta or TNF-alpha. These morphological alterations were associated with a significant rearrangement of the expression of mesothelial adhesion molecules as detected by flow cytometry. IL-1beta and TNF-alpha both led to a loss of the expression of the hemidesmosomal integrin subunits alpha6 ( P<0.01 and P<0.001) and beta4 ( P<0.01) and an increased expression of the integrin subunit alpha5 ( P<0.001 and P<0.01). IL-1beta furthermore upregulated the expression of the integrin subunits alpha1, alpha2 and the adhesion molecule CD44 while the latter was downregulated by TNF-alpha. Our data indicate that IL-1beta and TNF-alpha may significantly affect disease processes of the abdominal cavity by their potential to disrupt the mesothelial basal cell-matrix adhesion and, thus, the integrity of the peritoneal mesothelial cell lining.

Cell Adhesion Molecules↗
PubMed1998

Establishment of oral mucosa phenotype in vitro in correlation to epithelial anchorage.

Cell-matrix interactions and the ordered deposition of basement membrane (BM) components are of major importance for the maintenance of tissue homeostasis in complex epithelia. This aspect was studied in vitro in a coculture system designed as an oral mucosa model. As crucial epithelial features the kinetics of proliferation, expression of site-specific keratins as well as integrin patterns in correlation to synthesis of BM components were assessed by immunohistochemistry and in situ hybridization. Comparison with non-cornified gingiva as tissue of origin revealed different stages of epithelial development, eventually leading to complete reconstruction within a time frame of 1-3 weeks. First, the initial activated stage up to 1 week was characterized by (a) high keratinocyte proliferation, (b) extended expression of the basal cell-specific keratin K5 and (c) a patchy pattern of the differentiation-specific keratins K4 and K13. Second, after 2 weeks the improvement of histoarchitecture correlated to (a) predominant K5 expression in the basal and (b) extension of K4 and K13 within the suprabasal cell compartment, (c) high expression of integrins alpha3 beta1 and alpha6 beta4 including their ligand laminin-5 and (d) accumulating deposition of basement membrane components. Third, virtually complete tissue normalization at 3 weeks was indicated by (a) restriction of K5 to the basal cell area, (b) regular suprabasal localization of K4 and K13, (c) polarization of integrins to basal and parabasal cells and (d) linear codistribution of collagen IV, "classical" laminin (-1 or -10) and laminin-5 underneath the basal cells. Thus, these organotypic cocultures represent relevant equivalents for non-keratinized oral mucosa with typical gingival differentiation features and in addition suitable models for preclinical trials such as prospective dental material testing.

Cell Differentiation↗
PubMed2004

Bacterial heat shock protein 60 may increase epithelial cell migration through activation of MAP kinases and inhibition of alpha6beta4 integrin expression.

Exogenous heat shock proteins may modify cell behavior of infected epithelium. The effect of heat shock protein 60 (hsp60) of Actinobacillus actinomycetemcomitans and Escherichia coli, and human recombinant hsp60 on migration of HaCaT skin keratinocytes was studied using the Boyden chamber assay. Hsp60 from different species increased cell migration by two- to fivefold and this effect was inhibited by ERK inhibitor PD 98059, p38 inhibitor SB 203580, and a function-blocking epidermal growth factor receptor (EGFR) antibody. Hsp60 reduced the expression of alpha6-integrin mRNA and its protein levels on the cell surface but had no effect on the expression of beta4, beta1, alpha1, alpha5 or alphav integrin subunits. Hsp60 also significantly inhibited cell adhesion to laminin-5, a ligand of alpha6beta4 integrin. These results suggest that exogenous hsp60 released from bacteria or inflammatory cells may promote epithelial cell migration through activation of EGFR and MAP kinases, and inhibition of alpha6beta4 integrin expression.

Animals↗
PubMed1998

Age-related functional and structural changes in human dermo-epidermal junction components.

Cultured normal human keratinocytes obtained from 14 facial skin biopsies of donors aged 9-79 y were used to study the influence of donor age on the integrin receptors, cell adhesive properties in vitro, and type VII collagen synthesis. Immuno-spectrofluorimetric quantitation of integrins showed a decrease in the beta1- and beta4-subunits in low (0.08 mM) and high (1.8 mM) calcium conditions with aging. Calcium ions decreased the fluorescence intensity by relocating integrins at cell boundaries. Measurements of adhering cells showed that adhesion to bovine serum albumin-, type IV collagen- or laminin 1-coated plastic surfaces initially increased until donor age reached 30 y and then decreased. Specific adhesion to type IV collagen and laminin 1 did not vary with age, but the increase in adhesion to type IV collagen produced by manganese ions increased with age, suggesting an age-dependent feature of beta1 integrin. Synthesis of type VII collagen, increased or not by TGFbeta1 (10 ng per ml), did not vary with the donor age. Global normalized principal component analysis showed that variables related to integrins were strongly correlated, as were those of adhesion. Pre-embedding immunoelectron microscopy of freshly isolated keratinocytes showed that certain hemidesmosomes from aged cells had little or no reaction with anti-beta4-chain antibody. Post-embedding type IV collagen immunostaining and image analysis showed less type IV collagen in adult dermo-epidermal junctions. These findings indicate that there are structural and functional changes in the dermo-epidermal junction components with aging, probably giving a less effective epidermal anchoring system.

Adolescent↗
PubMed2001

Involvement of laminin binding integrins and laminin-5 in branching morphogenesis of the ureteric bud during kidney development.

Branching morphogenesis of the ureteric bud (UB) [induced by the metanephric mesenchyme (MM)] is necessary for normal kidney development. The role of integrins in this complex developmental process is not well understood. However, the recent advent of in vitro model systems to study branching of UB cells and isolated UB tissue makes possible a more detailed analysis of the integrins involved. We detected integrin subunits alpha3, alpha6, beta1, and beta4 in both the UB and cells derived from the early UB. Blocking the function of each of these integrin subunits individually markedly inhibited branching morphogenesis in cell culture models. However, inhibiting individual integrin function with blocking antibodies in whole kidney and isolated UB culture only partially inhibited UB branching morphogenesis, suggesting that, in these more complex in vitro systems, multiple integrins are involved in the branching program. In whole organ and isolated bud culture, marked retardation of UB branching was observed only when both alpha3 and alpha6 integrin subunits were inhibited. The alpha6 integrin subunit can be expressed as both alpha6beta1 and alpha6beta4, and both of these beta subunits are important for UB branching morphogenesis in both cell and organ culture. Furthermore, laminin-5, a common ligand for integrins alpha3beta1 and alpha6beta4, was detected in the developing UB and shown to be required for normal UB branching morphogenesis in whole embryonic kidney organ culture as well as isolated UB culture. Together, these data from UB cell culture, organ culture, and isolated UB culture models indicate that both integrin alpha3 and alpha6 subunits play a direct role in UB branching morphogenesis, as opposed to being modulators of the inductive effects of mesenchyme on UB development. Furthermore the data are consistent with a role for laminin-5, acting through its alpha3beta1 and/or alpha6beta4 integrin receptors, in UB branching during nephrogenesis. These data may help to partially explain the renal phenotype seen in integrin alpha3 and alpha3/alpha6 subunit-deficient animals.

Animals↗
PubMed1998

Endothelial cells assemble two distinct alpha6beta4-containing vimentin-associated structures: roles for ligand binding and the beta4 cytoplasmic tail.

The alpha6beta4 laminin binding integrin functions in the assembly of type I hemidesmosomes, which are specialized cell-matrix adhesion sites found in stratified epithelial cells. Although endothelial cells do not express all the components of type I hemidesmosomes, endothelial cells can express the alpha6beta4 integrin. Because endothelial cells lose expression of alpha6beta4 in culture, we expressed recombinant alpha6beta4 in the dermal microvascular endothelial cell line, HMEC-1, to test whether endothelial cells can assemble adhesion structures containing alpha6beta4. Using immunofluorescence microscopy, we found that recombinant alpha6beta4 concentrates specifically in a novel fibrillar structure on the basal surface of endothelial cells in the absence of an exogenous laminin substrate. This localization is regulated by an intracellular mechanism, because the beta4 cytoplasmic domain is sufficient to direct a reporter domain (IL-2R) to the fibrillar structures independently of recombinant alpha6beta4. In addition, this IL-2R-beta4 chimera is sufficient to recruit the intermediate filament-associated protein HD1/plectin to these fibrillar structures and this also occurs in the absence of recombinant alpha6beta4. The fibrillar localization pattern, as well as the recruitment of HD1/plectin, requires the first and second fibronectin type III repeats and the connecting segment of the beta4 tail. In addition, when endothelial cells are provided a laminin 5-rich matrix, recombinant alpha6beta4 redistributes from the fibrillar structure to type I hemidesmosome-like structures. The beta4 cytoplasmic domain can also direct a reporter domain to these type I hemidesmosome-like structures; however, this process is dependent upon the expression of recombinant alpha6beta4 Biochemical analysis indicates that both the fibrillar and the type I hemidesmosome-like structures are associated with the vimentin intermediate filament cytoskeleton. Thus, the results illustrate that endothelial cells have the essential components necessary to assemble at least two distinct alpha6beta4-containing and vimentin-associated structures on their basal surface and that the alpha6beta4 cytoplasmic tail and the availability of specific alph6beta4 ligands regulate receptor localization to these structures.

Antigens, Surface↗
PubMed2003

Temporal and spatial expression of integrins and their extracellular matrix ligands at the maternal-fetal interface in the rhesus monkey during pregnancy.

The integrin and extracellular matrix protein (ECM)-mediated adhesion and invasion of the receptive maternal uterine endometrium by trophoblasts is a critical event in the complex physiological process of pregnancy. Although the process has been largely characterized in mice, the relevant mechanism in primates remains unclear. We investigated the expression patterns and dynamic alterations of integrin subunits (alpha1, alpha5, alpha6, beta1, and beta4) and their ECM ligands, such as laminin (LN), type IV collagen (Col IV), and fibronectin (FN), at the maternal-fetal interface during Gestational Days 15, 25, 50, and 100 and at full term in 20 pregnant rhesus monkeys. Immunohistochemistry and in situ hybridization revealed that a relatively high expression of integrins occurred in trophoblast cells at Gestational Day 15, with the peak level occurring at Day 25. The expression level decreased from Day 50 to term. Along the invasive pathway, expression levels of integrin alpha1, alpha5, and beta1 subunits were gradually elevated from the proximal to distal column, reaching peak level in the trophoblast shell, but were reduced in those invasive extravillous cytotrophoblast (EVCT) cells in contact with the decidua. Integrin alpha1, alpha5, beta1, and beta4 subunits were also highly expressed in decidual stromal cells and moderately expressed in the maternal epithelium and endothelium. Immunoreactive FN, LN, and Col IV were distributed in EVCT and decidual stromal cells and part of the uterine epithelial and endothelial cells. These data suggest that the correlated expression of integrins and their ECM ligands at the maternal-fetal interface might be involved in regulation of cell proliferation and differentiation and the counterbalanced invasion-accelerating and invasion-restraining processes in trophoblast cells during the early stage of pregnancy.

Animals↗
PubMed1996

The intracellular functions of alpha6beta4 integrin are regulated by EGF.

Upon ligand binding, the alpha6beta4 integrin becomes phosphorylated on tyrosine residues and combines sequentially with the adaptor molecules Shc and Grb2, linking to the ras pathway, and with cytoskeletal elements of hemidesmosomes. Since alpha6beta4 is expressed in a variety of tissues regulated by the EGF receptor (EGFR), we have examined the effect of EGF on the cytoskeletal and signaling functions of alpha6beta4. Experiments of immunoblotting with anti-phosphotyrosine antibodies and immunoprecipitation followed by phosphoamino acid analysis and phosphopeptide mapping showed that activation of the EGFR causes phosphorylation of the beta4 subunit at multiple tyrosine residues, and this event requires ligation of the integrin by laminins or specific antibodies. Immunoprecipitation experiments indicated that stimulation with EGF does not result in association of alpha6beta4 with Shc. In contrast, EGF can partially suppress the recruitment of Shc to ligated alpha6beta4. Immunofluorescent analysis revealed that EGF treatment does not induce increased assembly of hemidesmosomes, but instead causes a deterioration of these adhesive structures. Finally, Boyden chamber assays indicated that exposure to EGF results in upregulation of alpha6beta4-mediated cell migration toward laminins. We conclude that EGF-dependent signals suppress the association of activated alpha6beta4 with both signaling and cytoskeletal molecules, but upregulate alpha6beta4-dependent cell migration. The changes in alpha6beta4 function induced by EGF may play a role during wound healing and tumorigenesis.

Adaptor Proteins, Signal Transducing↗
PubMed2003

Modulation of keratin and connexin expression in limbal epithelium expanded on denuded amniotic membrane with and without a 3T3 fibroblast feeder layer.

PURPOSE: Based on the knowledge that limbal epithelial stem cells (SCs) do not express keratin-3 (K3), connexin (Cx)43, and Cx50, a study was conducted to investigate amniotic membrane (AM) culturing conditions that promote limbal SC expansion. METHODS: Human limbal epithelium was expanded on intact and epithelially denuded AM, with or without a 3T3 feeder layer, and subsequently transplanted to nude mice to induce epithelial stratification and differentiation. Immunostaining and Western blot analysis were used to determine protein expression of K3, Cx43, and Cx50. Expression of integrin-alpha3, -beta1, -alpha6, and -beta4 was investigated by immunostaining. RESULTS: Protein levels of K3, Cx43, and Cx50 in limbal epithelium on intact AM was lower than those on denuded AM. Addition of 3T3 to denuded AM increased the level of Cx43 but decreased that of Cx50. After xenotransplantation, the basal layer of the resultant stratified epithelium on intact AM did not express K3, Cx43, and Cx50, whereas that on denuded AM expressed all three markers. The addition of 3T3 resulted in positive staining of Cx43 and K3 but negative staining of Cx50 in the basal epithelium. After stratification, integrin expression was detected at the basal epithelium-amniotic basement membrane interface in all three culture conditions. CONCLUSIONS: Limbal cultures on intact AM retain a limbal epithelial phenotype, whereas those on denuded AM differentiate into a corneal phenotype. The addition of 3T3 slows but does not prevent corneal differentiation on denuded AM. Such a difference may involve integrin-mediated extracellular matrix interactions.

3T3 Cells↗